carbamoyl tetrazoles as inhibitors of endocannabinoid inactivation: a critical revisitation

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European Journal of Medicinal Chemistry 43 (2008) 62e72http://www.elsevier.com/locate/ejmech

Original article

Carbamoyl tetrazoles as inhibitors of endocannabinoidinactivation: A critical revisitation

Giorgio Ortar a, Maria Grazia Cascio b,c, Aniello Schiano Moriello b, Mercedes Camalli d,Enrico Morera a, Marianna Nalli a, Vincenzo Di Marzo b,*

a Dipartimento di Studi Farmaceutici, Universita di Roma ‘La Sapienza’, piazzale Aldo Moro 5, 00185 Roma, Italyb Endocannabinoid Research Group, Institute of Biomolecular Chemistry, Consiglio Nazionale delle Ricerche,

via dei Campi Flegrei 34, 80078 Pozzuoli (Napoli), Italyc Dipartimento di Scienze Farmaceutiche, Universita degli Studi di Salerno, via Ponte Don Melillo, 84084 Fisciano (SA), Italy

d Istituto di Cristallografia, Consiglio Nazionale delle Ricerche, Area della Ricerca Roma 1, 00016 Monterotondo Stazione, Roma, Italy

Received 24 November 2006; accepted 28 February 2007

Available online 19 March 2007

Abstract

We have synthesized a series of 18 1,5- and 2,5-disubstituted carbamoyl tetrazoles, including LY2183240 (1) and LY2318912 (7), twocompounds previously described as potent inhibitors of the cellular uptake of the endocannabinoid anandamide, and their regioisomers 2 and 8.We confirm that compound 1 is a potent inhibitor of both the cellular uptake and, like the other new compounds synthesized here, the enzymatichydrolysis of anandamide. With the exception of 9, 12, 15, and the 2,5-regioisomer of LY2183240 2, the other compounds were all found to beweakly active or inactive on anandamide uptake. Several compounds also inhibited the enzymatic hydrolysis of the other main endocannabinoid,2-arachidonoylglycerol, as well as its enzymatic release from sn-1-oleoyl-2-arachidonoyl-glycerol, at submicromolar concentrations. Four of thenovel compounds, i.e. 3, 4, 17, and 18, inhibited anandamide hydrolysis potently (IC50¼ 2.1e5.4 nM) and selectively over all the other targetstested (IC50� 10 mM), thus representing new potentially useful tools for the inhibition of fatty acid amide hydrolase.� 2007 Elsevier Masson SAS. All rights reserved.

Keywords: Endocannabinoids; Anandamide cellular uptake inhibitors; Fatty acid amide hydrolase inhibitors; Carbamoyl tetrazoles

1. Introduction

Endocannabinoids, i.e. endogenous agonists of CB1 andCB2 cannabinoid receptors [1,2], are involved in an extraordi-narily large number of physiological and pathological condi-tions in mammals [3,4]. Increasing evidence suggests thatthe modulation of endocannabinoid levels might be of valu-able therapeutic relevance in the treatment of a variety of path-ological conditions including anorexia, anxiety, inflammation,nausea and emesis, neuropsychiatric disorders, pain and spas-ticity [5]. The levels of the two main endocannabinoids, anan-damide (AEA) [6] and 2-arachidonoylglycerol (2-AG) [7,8],

* Corresponding author. Tel.: þ39 081 8675093; fax: þ39 081 8041770.

E-mail address: [email protected] (V. Di Marzo).

0223-5234/$ - see front matter � 2007 Elsevier Masson SAS. All rights reserved.

doi:10.1016/j.ejmech.2007.02.023

are regulated by specific biosynthetic and degradative path-ways, most of which have been now fully characterized andthe relevant enzymes cloned [9]. The termination of the actionof AEA is regulated by its cellular uptake, followed by intra-cellular hydrolysis by fatty acid amide hydrolase (FAAH). Themechanism of AEA transport, first characterized in 1994 by DiMarzo et al. [10], has been proposed to be an ATP-indepen-dent process of facilitated diffusion. However, a protein ableto specifically bind AEA and facilitate its migration acrossthe cell membrane has not yet been identified, its existenceis still controversial, and alternative hypotheses have been pro-posed, including passive diffusion, endocytosis, and intracellu-lar sequestration [11e17]. Much of the discussion in recentyears has been centred on the issue of whether or not AEAuptake is driven uniquely by the FAAH-catalyzed hydrolysisof the compound.

mailto:[email protected]

http://www.elsevier.com/locate/ejmech

N NN

N

N

O

N NN

N

N

OR

O

N NN

NNH

N

OI

R

O

N NN

NNH

N

O

N NN

NN

O

N NN

NR

NO

N NN

NNH

NO

O

N3N3

I

N NN

NNH

NO

R

O

3: R = PhCH24: R = Ph(CH2)2

5: R = PhCH26: R = Ph(CH2)2

9: R = 3-biphenyl10: R = 4-biphenyl11: R = 4-biphenylmethyl12: R = Ph(CH2)613: R = Ph(CH2)7

1 (LY2183240) 2

7 (LY2318912) 8


Fig. 1. Structures of carbamoyl tetrazoles synthesized and tested in this study.

63G. Ortar et al. / European Journal of Medicinal Chemistry 43 (2008) 62e72

While the nature of the mechanism responsible for AEAuptake is still a matter of debate, a large number of arachi-donic or oleic acid-based compounds that effectively inhibitthis process have been described over the last decade.Although the ability of many of them to inhibit also FAAHhas hampered the accurate characterization of their selectivityfor either target, the recent individuation of novel selectiveAEA transport inhibitors that do not appear to inhibit FAAHsuch as UCM707 [18], AM1172 [13] and OMDM1,2 [19]has stimulated further studies to distinguish between the rela-tive contributions of the putative AEA transporter and FAAHin AEA removal process.

From a medicinal chemistry perspective, the identificationof potent and selective inhibitors of AEA transport lackinga long acyl side chain would be advantageous in terms ofmore precise target identification and of their potential of be-ing made into therapeutic drugs. In this respect, Hopkins andWang reported in 2004 in abstract form a non-arachidonoylcompound, SEP-0200228 that inhibited AEA uptake in humanmonocytes with an IC50 of 90 nM [20]. However, a full publi-cation of this study has not yet appeared. More recently,Moore et al. described a potent, competitive small moleculeinhibitor of AEA uptake, LY2318912, which has allowed theidentification of a high-affinity AEA binding site distinctfrom FAAH in RBL-2H3 cell plasma membranes [21]. Theyalso tested the effect of the parent compound of LY2318912,LY2183240, in a pharmacological test of persistent pain.

As underlined by Mechoulam and Deutsch in the commen-tary accompanying Moore et al.’s paper [22], ‘the identifica-tion of a high-affinity binding site involved in the transportof AEA by Eli Lilly group represents a major step in under-standing endocannabinoid metabolism and will present drugresearchers with a very valuable tool’. However, these authorsalso raised a number of questions regarding the possibility thatthe novel compounds developed at Eli Lilly might act viaadditional mechanisms other than inhibition of AEA cellularuptake. Indeed, the Eli Lilly group subsequently reported thatLY2183240 does inhibit enzyme activity in purified FAAHpreparations [23], whereas very recently Alexander and Cravatthave shown that LY2183240 is a potent, covalent inhibitor notonly of FAAH but also of several other serine proteases, includ-ing the monoacylglycerol lipase (MAGL) that catalyses thehydrolysis of 2-arachidonoylglycerol [24].

The compound described by Alexander and Cravatt is,however, in all evidence a mixture of regioisomeric 1,5- and2,5-disubstituted tetrazoles (see Section 2), while neither de-tails of the syntheses of LY2183240 and LY2318912 nor anyinformation on the activity of their 2,5-regioisomers hasbeen presented in the Eli Lilly papers. This despite the factthat the main procedure for the preparation of disubstitutedtetrazoles, i.e. the alkylation (arylation, acylation) of 5-substituted tetrazoles, affords generally mixtures of regio-isomers and difficulties are often encountered in their separationand/or when assigning their regiochemistry [25].

In light of these uncertainties of both synthetic and bio-logical nature and in an effort to gain further insight intothe actions of tetrazolic ureas, in particular the individual

regioisomers, we have synthesized a series of nine regioisomericcouples of 1,5- and 2,5-disubstituted carbamoyl tetrazoles,including LY2183240, LY2318912 and their regioisomers(Fig. 1), by varying the substituent in the 5-position, andhave investigated the effects of the regiochemistry and ofthe 5-substitution on the activity of these compounds onboth AEA uptake and FAAH. The capability of these com-pounds to also inhibit the MAGL as well as the diacylglycerollipase catalysing the biosynthesis of 2-AG [3] has also beenassessed.

2. Chemistry

The tetrazole compounds in Fig. 1 were prepared as de-tailed below and as summarized in Schemes 1e3, followingsome of the newer methods for 5-substituted tetrazole

N NN

NRCNR 1 + 2 (or 3 + 5 or 4 + 6)

a

H19: R = 4-biphenyl20: R = CH2Ph21: R = (CH2)2Ph

22: R = 4-biphenyl23: R = CH2Ph24: R = (CH2)2Ph

b

Scheme 1. Synthesis of tetrazoles 1e6. Reagents and conditions: (a) n-Bu3SnN3, dioxane, reflux, 20 h, then dry HCl, rt, 15 min; (b) Me2NCOCl, Et3N, MeCN,

rt, 15 h.

64 G. Ortar et al. / European Journal of Medicinal Chemistry 43 (2008) 62e72

formation from organic nitriles [26]. Refluxing the commer-cially available 4-biphenylacetonitrile 19 with tri-n-butyltinazide [27], followed by an acidic hydrolysis step to removethe tin group from the tetrazole ring and acylation of theresulting tetrazole 22 [28] with N,N-dimethylcarbamoyl chlo-ride gave a 0.58:1 mixture of 1,5- and 2,5-regioisomers 1(LY2183240) and 2 which could be separated by silica gelchromatography, the less polar 2,5-disubstituted tetrazole 2,eluting prior to the 1,5-disubstituted tetrazole 1 (Scheme 1).A mixture of regioisomers was also obtained using the sameconditions employed by Alexander and Cravatt [24] for thecarbamoylation of 22 and, indeed, the 1H NMR spectrumreported by them for the compound assumed to be pure 1was the sum of those of 1 and 2.

The structures of the regioisomeric tetrazoles were first ten-tatively assigned on the basis of 13C NMR spectra. 13C NMRstudies of disubstituted tetrazoles have shown that the CN4

carbon atom of 1,5-disubstituted tetrazoles is more shieldedthan the corresponding carbon atom of the 2,5-disubstitutedtetrazoles [29e31], but exceptions to this general rule canbe found [32]. Based on these reported observations, the tetra-zole which was first eluted was designated the 2,5-disubsti-tuted tetrazole 2, since it showed a CN4

13C resonance atd 164.99 ppm, while the more polar tetrazole 1 had a CN413C resonance at d 155.53 ppm. However, more spectroscopicevidence for the regiochemical assignment of the tworegioisomers could not be obtained by a heteronuclear multi-ple bond correlation (HMBC) experiments, since both the

ICO2Me

H2N H2N

I

a

c d,e

OI

N NN

NNH

H

g

7

25 26

28

30

CO2H

N3

N3

Scheme 2. Synthesis of tetrazoles 7 and 8. Reagents and conditions: (a) ICl, AcOH,

15 min; (c) 1 M LiOH, THF/H2O, rt, 15 h; (d) SOCl2, 100 �C, 1 h; (e) H2NCH2CN

HCl, rt, 15 min; (h) Me2NCOCl, Et3N, MeCN, rt, 15 h.

compounds showed similar HMBC correlations. The 2,5-di-substituted structure of 2 was thus unambiguously determinedby X-ray crystal structural analysis. Perspective view of 2 withatom numbering is shown in Fig. 2, while crystal data col-lection procedures, structure determination methods and re-finement results are summarized in Table 1.

Repetition of the above procedure on nitriles 20 and 21 fur-nished similar and separable mixtures of 1,5- and 2,5-re-gioisomers 3þ 5 and 4þ 6, respectively.

As concerns the preparation of regioisomers 7(LY2318912) and 8, methyl 4-aminobenzoate (25) was con-verted into methyl 4-amino-3-iodobenzoate (26) according toSpivey et al. [33]. Reaction of 26 with sodium nitrite andsodium azide in acidic medium followed by alkaline hydrolysisof the ester 27 as described previously [34] afforded 4-azido-3-iodobenzoic acid (28). Reaction of the acyl chloride derivedfrom 28 by the action of SOCl2 with aminoacetonitrile bisul-fate furnished the amide 29. Refluxing of 29 with tri-n-butyltinazide followed by acidic hydrolysis and acylation of tetrazole30 with N,N-dimethylcarbamoyl chloride afforded a 0.52:1mixture of 1,5- and 2,5-regioisomers 7 and 8 which could beseparated by silica gel chromatography, again the less polar2,5-disubstituted tetrazole 8 eluting prior to the 1,5-disubsti-tuted tetrazole 7 (Scheme 2).

Repetition of the three-step procedure on acids 31e35furnished mixtures of 1,5- and 2,5-regioisomers which couldbe separated in the case of tetrazoles 9þ 14, 12þ 17 and13þ 18, while from the mixture of regioisomers 10þ 15

I CO2MeCO2Meb

I NH

CN

O

f

+ 8

27

29

N3

N3

rt, 1 h; (b) 4 N HCl, NaNO2, AcOH/H2O, 0e5 �C, 15 min, then NaN3, H2O, rt,

$H2SO4, Et3N, CH2Cl2, rt, 15 h; (f) n-Bu3SnN3, dioxane, reflux, 20 h, then dry

CO2Hb

c


a

R NH

CN

OR N

H

O

N NN

N

H



9 + 14 (or 10 + 15 or 11 + 16 or 12 + 17 or 13 + 18)

R

Scheme 3. Synthesis of tetrazoles 9e18. Reagents and conditions: (a) HOBt/EDC, rt, 1 h, then H2NCH2CN$H2SO4, Et3N, CH2Cl2, rt, 15 h; (b) n-Bu3SnN3,

dioxane, reflux, 20 h, then dry HCl, rt, 15 min; (c) Me2NCOCl, Et3N, MeCN, rt, 15 h.


only 2,5-regioisomer 15 could be isolated in a pure form,owing to the propensity of 1,5-regioisomer 10 to isomeriseduring chromatographic separation, and attempts to separateregioisomers 11þ 16 were unsuccessful (Scheme 3).

Table 1

Crystal data of compound 2

Empirical formula C17H17N5O

Formula weight 307.36

Crystal size (mm) 0.4� 0.3� 0.3

Z 4

Density (calculated) (g/cm3) 1.294

Crystal system Monoclinic

Space group P21/c

a (A) 6.112 (4)

b (A) 8.907 (4)

c (A) 29.07 (2)

b (�) 94.27 (9)

3. Biological evaluation

Apart from testing for the first time 13 new compounds(two of which as a mixture of regioisomers) and the 2,5-isomers 2, 8 of the Eli Lilly compounds, whose biologicalactivity was not reported in Moore et al.’s paper [21], we havealso re-examined in this study the activity of the previouslydescribed 1,5-isomers 1, 7, using different conditions for theassay of [14C]anandamide cellular uptake. These conditions,by using higher concentrations of the [14C]anandamide substrate,have proved in the past to be more restrictive than other assaysregarding potency and/or efficacy of competitive uptake inhib-itors [35] and, by using incubation times with intact rat baso-philic leukemia RBL-2H3 cell that are shorter (90 s) thanthose used by Moore et al., minimize the interference ofFAAH inhibition by the compounds on their net effect on[14C]anandamide uptake [11]. We have also used a 10 minpre-incubation of cells with the compound to maximize theirpotential competition with the specific site(s) involved inanandamide cellular uptake. In view of the results published

Fig. 2. ORTEP projection of the tetrazole 2.

by Alexander and Cravatt, showing that carbamoyl tetrazolesact as inhibitors for several serine hydrolases, includingFAAH and MAGL [24], we have used membranes from ratbrain to assess the FAAH inhibitory activity. This method isnow to be considered even more suitable than in the past sincerecent data indicate that in some mammals, but not in rats ormice, a second FAAH isoform (FAAH-2) is also abundantlyexpressed [36]. In order to determine whether the compoundswould also act as inhibitors of 2-AG hydrolysis via theMAGL, and of 2-AG biosynthesis via the sn-1-selective diac-ylglycerol lipases (DAGLs), we have used the cytosolic andmembrane fractions, respectively, of COS cells overexpressingDAGLa, since COS cells exhibit high levels of a cytosolic

Volume (A3) 1578.2 (17)

Wavelength (A) 1.54178

Temperature (K) 293

Reflections number (total) 2483

Reflections number (observed) 1946

Reflections threshold expression F> 3.0sjFjDiffraction measurement method we2w

qmin, qmax 1.50e31.04

m (mm�1) 0.698

Refine number parameters 208

R factor 0.065

wR factor 0.083

Goodness of fit 0.88

Shift/error-max 0.000

Shift/su_mean 0.000

Final e.s.d. of r 0.64

Computing structure solution SIR200a

Computing structure refinement SIR2004a

a Ref. [39].


constitutive MAGL activity whereas their overexpressed DAGLactivity is concentrated in the membranes [37]. Only 15 ofthe 18 compounds could be tested in a regioisomerically pureform. Compound 10 was unstable and easily got converted to15, and therefore could not be tested. The two regioisomers11 and 16 were tested in mixture, as they could not be separatedchromatographically.

4. Results and discussion

The results of the pharmacological assays shown in Table 2can be summarized as follows: (1) we could confirm that com-pound 1 is a potent inhibitor of both the cellular uptake and theenzymatic hydrolysis of anandamide [21,24]; (2) with theexception of 9, 12, 15, and the 2,5-regioisomer of LY2183240(2), which exhibited lower potency than 1, but were still capa-ble of efficaciously inhibiting anandamide cellular uptake withIC50 values between 0.1 and 1 mM, the other compounds wereall weakly active or inactive in this assay; (3) all compoundsinhibited anandamide hydrolysis by rat brain membraneswith IC50 values ranging from 2.1 to 33 nM; (4) several com-pounds also inhibited the enzymatic hydrolysis of the otherendocannabinoid, 2-AG, as well as its enzymatic releasefrom sn-1-oleoyl-2-arachidonoyl-glycerol, at submicromolarconcentrations; (5) four of the novel compounds, i.e. 3, 4,17, and 18, inhibited anandamide hydrolysis potently (IC50¼2.1e5.4 nM) and with high (500e2000-fold) selectivity overall the other targets tested.

Table 2

Effect of the compounds synthesized in this study on [14C]anandamide hydro-

lysis by rat brain membranes (which express FAAH as the only anandamide

hydrolysing enzyme), [14C]anandamide uptake by intact RBL-2H3 cells

(where a putative anandamide transporter has been characterized pharma-

cologically), [14C]2-AG hydrolysis by COS cell cytosolic fractions (which

express MAGL), and sn-1-[14C]oleoyl-2-arachidonoyl-glycerol (DAG) conver-

sion into 2-AG and [14C]oleic acid by COS cells membranes overexpressing

human recombinant DAGLaa

Compound AEA

hydrolysis

AEA

uptake

2-AG

hydrolysis

DAG hydrolysis

to 2-AG

1 0.0021 0.015 8.10 >10

2 0.0330 0.998 0.02 0.51

3 0.0054 >10 (15.8%) 10 10

4 0.0027 >10 (18.0%) 10 10

5 0.0035 >10 (10.9%) 0.48 0.60

6 0.0032 >10 (16.0%) 0.11 0.50

7 0.0230 >1 (32.4%) 0.35 0.13

8 0.0250 >1 (9.0%) >10 4.40

9 0.0320 0.090 0.85 0.34

11þ 16 0.0290 >10 (35.2%) 0.10 1.00

12 0.0037 0.851 0.02 0.20

13 0.0100 >10 (37.5%) 0.04 0.57

14 0.0230 >10 (23.0%) 10 >10

15 0.0060 0.985 >10 >10

17 0.0021 >10 (30.0%) >10 10

18 0.0032 >10 (18.8%) 10 10

a Data are expressed as IC50 (mM) and in the case of weak inhibitors as the

maximal percent inhibition observed (between parentheses), and are means of

n¼ 3e4 separate determinations. Standard errors are not shown for the sake of

clarity and were never higher than 10% of the means.

Although we have confirmed here that the most potentanandamide uptake inhibitor identified by Moore et al., i.e.compound 1, is also the most potent compound in this assayunder our conditions, we could not confirm here the samepotency (IC50¼ 0.27 nM, i.e. w55-fold higher) published pre-viously for this compound. The discrepancy is even moreevident with compound 7, which was previously reported toinhibit AEA uptake in the same cells with an IC50 of 7.2 nMand was found here to exhibit w140-fold lower potency.The exact reasons for these differences are not known, butare very likely due to the fact that we used experimental con-ditions different from those used in the previous study (namelymuch shorter incubation times and higher concentrations ofsubstrate [14C]anandamide).

Furthermore, as predicted by Mechoulam and Deutsch [22],and later shown by both the Eli Lilly group [23] and Alexanderand Cravatt [24], we found that all compounds also potentlyinhibit rat brain FAAH. However, additional data also indicatethat the effect of compound 1 on AEA uptake is only in partdue to its capability of inhibiting AEA hydrolysis. First,we observed that under the incubation conditions used here,the extent of temperature-sensitive [14C]anandamide uptake(16.2� 3.2% of total incubated anandamide) was significantlyhigher than that of [14C]anandamide hydrolysis (1.9� 0.1% oftotal incubated anandamide, means� SD of N¼ 10). Further-more, when this was tested in the same experiment in intactRBL-2H3 cells, compound 1 inhibited the uptake of[14C]anandamide more efficaciously than it inhibited the for-mation of [14C]ethanolamine from [14C]anandamide (respec-tively, 66.5� 11.4 and 74.2� 8.1% inhibition of uptake and41.1� 6.1 and 36.7� 5.0% inhibition of hydrolysis at 0.1and 1 mM, respectively, p< 0.01 in both cases, N¼ 4). Thesefindings suggest that, in intact cells, compound 1 inhibitsanandamide hydrolysis, which is an intracellular process,because it inhibits anandamide uptake and not vice versa.Thus, although part of the inhibitory effect on anandamideuptake by compound 1 is clearly due to its inhibition ofFAAH, which contributes to driving cellular uptake, this com-pound seems capable of influencing the former process also ina way independent from anandamide hydrolysis. This con-clusion is also supported by the data recently published byDickason-Chesterfield et al., who also found a clear dissocia-tion between the FAAH- and anandamide uptake-inhibitoryactivities of several compounds, including compound 1 [23].

As could be predicted again by the study by Alexander andCravatt [24], several of the carbamoyl tetrazoles synthesized inthis study also potently inhibited the hydrolysis of [14C]2-AGby a MAGL-like activity present in the cytosol of COS cells.Furthermore, we showed here for the first time that these com-pounds can also be good inhibitors of 2-AG biosynthesis fromsn-1-[14C]oleoyl-2-arachidonoyl-glycerol. However, we agreeonly in part with the conclusions drawn by these authorsthat ‘the promiscuity of LY2183240 designates the heterocy-clic urea as a chemotype with potentially excessive proteinreactivity for drug design’. In fact, we did find that out of the17 tetrazoles screened, four compounds exhibited potent activ-ity for FAAH selectively over all other targets tested here.


These compounds, i.e. 3, 4, 17, and 18, might represent newuseful FAAH inhibitors exploitable for therapeutic use inpathologies as diverse as those listed in Section 1.

Our results in 12 carbamoyl tetrazoles, which represent six1,5- and 2,5-regioisomeric couples, together with thoseobtained with 1 and 7 and their 2,5-regioisomers 2 and 8 allowus to delineate for the first time some structureeactivity rela-tionships for the interactions of this class of compounds withthe putative anandamide membrane transporter, FAAH,MAGL and DAGLa, and hence provide new information forthe future design of new pharmacological tools interactingwith these targets. In particular, the following conclusionscan be drawn from the data in Table 2: (1) regarding the inhi-bition of anandamide cellular uptake, most 1,5-isomers aresignificantly more potent than the corresponding 2,5-isomers.Furthermore, conformationally less flexible substituents in the5-position seem to yield better inhibitors than more flexibleones. In any case, the features of 5-substitution necessary forpotent uptake inhibition appear to be rather stringent, as seem-ingly minor structural differences produce remarkable varia-tions of the activity (e.g. compounds 14, 15 and 12, 13); (2)by contrast, 2,5-isomers can also be more potent FAAH inhib-itors than their 1,5-counterparts; the requirements of the 5-sub-stitution are less critical and conformationally more flexiblesubstituents appear to afford more potent inhibitors. Theseobservations represent yet another indirect piece of evidencein favour of the existence of a FAAH-independent mechanismfor anandamide uptake; (3) regarding the inhibition of MAGLand DAGL, 1,5-isomers were significantly more potent thanthe corresponding 2,5-isomers in the case of compoundswith an amide function in the 5-side chain, while the oppositeseems to apply for compounds with non-functionalized sidechains; (4) with a few exceptions, carbamoyl tetrazoles seemto be significantly more potent at inhibiting MAGL thanDAGL. Nevertheless, this property is not likely to be exploitedfor the development of selective MAGL inhibitors since themost potent inhibitors of 2-AG hydrolysis synthesized in thepresent study were as potent as FAAH inhibitors.

5. Conclusions

We have provided here new methods for the syntheses ofcarbamoyl tetrazoles with activity as inhibitors of endocanna-binoid inactivation, as well as new data on the structureeactivity relationships for the interactions of these compoundswith the putative anandamide membrane transporter, FAAH,MAGL and DAGLa. Our data provide further indirect evi-dence that a specific and FAAH-independent process underliespart of the cellular uptake of anandamide by RBL-2H3 cells,but argue against the possibility of developing selective inhib-itors of such process from carbamoyl tetrazoles. On the otherhand, we have shown here that it might be possible to developselective as well as potent FAAH inhibitors from the chemicalmodification of the compounds previously reported by EliLilly. Four such new inhibitors i.e. 3, 4, 17, and 18, havebeen described here.

6. Experimental protocols

6.1. Chemistry

All chemical reagents were commercially available unlessotherwise indicated. Melting points were determined on a Koflerhot-stage apparatus and are uncorrected. IR spectra were re-corded on a Perkin-Elmer 1000 FT-IR spectrophotometer asKBr disks unless otherwise indicated. 1H and 13C NMR spectrawere obtained on a Varian Mercury 300 spectrometer usingCDCl3 as solvent unless otherwise indicated and TMS as internalstandard. Analyses indicated by the symbols of the elements orfunctions were within�0.4% of the theoretical values (Table 3).

Compounds 26 [33], 27 [34], 28 [34], and 31 [38] havebeen described in the literature.

6.1.1. 5-((Biphenyl-4-yl)methyl)-1H-tetrazole (22)A solution of 4-biphenylacetonitrile (19) (1.449 g, 7.5 mmol)

and n-Bu3SnN3 [27] (3.736 g, 11.25 mmol) in 1,4-dioxane(15 mL) was refluxed for 20 h, cooled at room temperature,and gaseous HCl was added over 15 min. The mixture wasdiluted with water, neutralized with 2 N NaOH, and extractedwith AcOEt. The organic phase was washed with water, dried(Na2SO4), and evaporated under vacuum to leave a residue(5.20 g) which was triturated with hot hexane. The resultingsolid was filtered off, washed with hexane, and dried undervacuum to give 1.460 g (82%) of 22. Mp 215e217 �C; IR3117, 3029, 2853, 2727, 2623, 1575, 1487, 1424, 1256,1106, 1053 cm�1; 1H NMR (300 MHz) d 4.37 (2H, s), 7.36e7.67 (9H, m); 13C NMR (75 MHz) d 28.53, 126.53, 126.94,127.37, 128.85, 129.21, 135.05, 138.93, 139.69, 155.17. Anal.(C14H12N4) C, H, N.

6.1.2. 5-((Biphenyl-4-yl)methyl)-N,N-dimethyl-1H-tetrazole-1-carboxamide (1) and 5-((biphenyl-4-yl)methyl)-N,N-dimethyl-2H-tetrazole-2-carboxamide (2)

To a stirred solution of 22 (1.460 g, 6.18 mmol) and Et3N(1.73 mL, 12.36 mmol) in acetonitrile (10 mL) Me2NCOCl(0.85 mL, 9.27 mmol) was added at 0 �C, and the mixturewas stirred at 0 �C for 15 h. The mixture was diluted withwater and extracted with AcOEt. The organic phase waswashed twice with brine, dried (Na2SO4), and evaporatedunder vacuum. The residue (1.91 g) was chromatographedon silica gel (200 g) with CH2Cl2/AcOEt¼ 99/1 as eluent togive 2 (1.041 g, 55%) followed by 1 (609 mg, 32%). Com-pound 2: mp 80e81 �C; IR (CHCl3) 3010, 1732, 1488,1396, 1257, 1231, 1088 cm�1; 1H NMR (300 MHz) d 3.11(3H, s), 3.25 (3H, s), 4.37 (2H, s), 7.33e7.58 (9H, m); 13CNMR (75 MHz) d 31.26, 37.98, 39.00, 126.89, 127.14,127.35, 128.60, 129.16, 134.78, 140.00, 140.55, 147.66,164.99. Anal. (C17H17N5O) C, H, N. Compound 1: mp 87e88 �C; IR (CHCl3) 3011, 1742, 1489, 1393, 1235, 1166,1130, 1068 cm�1; 1H NMR (300 MHz) d 2.69 (3H, s), 3.07(3H, s), 4.48 (2H, s), 7.32e7.57 (9H, m); 13C NMR(75 MHz) d 29.43, 37.22, 38.57, 126.75, 127.26, 127.37,128.66, 129.26, 133.07, 140.07, 140.46, 147.71, 155.53.Anal. (C17H17N5O) C, H, N.

Table 3

Elemental analysis data

Compound Molecular formula Calculated C, H, N Found C, H, N

1 C17H17N5O C, 66.43; H, 5.58; N, 22.79 C, 66.49; H, 5.49; N, 22.65

2 C17H17N5O C, 66.43; H, 5.58; N, 22.79 C, 66.37; H, 5.62; N, 22.87

3 C12H15N5O C, 58.76; H, 6.16; N, 28.55 C, 58.59; H, 6.31; N, 28.67

4 C13H17N5O C, 60.21; H, 6.61; N, 27.01 C, 60.37; H, 6.44; N, 26.83

5 C12H15N5O C, 58.76; H, 6.16; N, 28.55 C, 59.02; H, 6.03; N, 28.18

6 C13H17N5O C, 60.21; H, 6.61; N, 27.01 C, 60.40; H, 6.47; N, 27.33

7 C12H12IN9O2 C, 32.67; H, 2.74; N, 28.57 C, 32.72; H, 2.72; N, 28.57

8 C12H12IN9O2 C, 32.67; H, 2.74; N, 28.57 C, 32.59; H, 2.70; N, 28.49

9 C18H18N6O2 C, 61.70; H, 5.18; N, 23.99 C, 61.48; H, 5.09; N, 24.08

12 C18H26N6O2 C, 60.32; H, 7.31; N, 23.45 C, 60.14; H, 7.28; N, 23.47

13 C19H28N6O2 C, 61.27; H, 7.58; N, 22.56 C, 61.03; H, 7.49; N, 22.48

14 C18H18N6O2 C, 61.70; H, 5.18; N, 23.99 C, 61.54; H, 5.07; N, 24.16

15 C18H18N6O2 C, 61.70; H, 5.18; N, 23.99 C, 61.67; H, 5.19; N, 23.90

17 C18H26N6O2 C, 60.32; H, 7.31; N, 23.45 C, 59.97; H, 7.44; N, 23.22

18 C19H28N6O2 C, 61.27; H, 7.58; N, 22.56 C, 61.30; H, 7.67; N, 22.67

22 C14H12N4 C, 71.17; H, 5.12; N, 23.71 C, 71.39; H, 5.18; N, 23.64

23 C9H10N4 C, 62.05; H, 5.79; N, 32.16 C, 62.16; H, 5.65; N, 32.47

24 C10H12N4 C, 63.81; H, 6.43; N, 29.77 C, 63.58; H, 6.19; N, 29.75

29 C9H6IN5O C, 33.05; H, 1.85; N, 21.41 C, 32.89; H, 1.97; N, 21.37

30 C9H7IN8O C, 29.21; H, 1.91; N, 30.28 C, 29.36; H, 1.87; N, 30.35

36 C15H12N2O C, 76.25; H, 5.12; N, 11.86 C, 76.47; H, 5.07; N, 11.68

37 C15H12N2O C, 76.25; H, 5.12; N, 11.86 C, 76.32; H, 5.02; N, 12.01

38 C16H14N2O C, 76.78; H, 5.64; N, 11.19 C, 76.68; H, 5.60; N, 11.13

39 C15H20N2O C, 73.74; H, 8.25; N, 11.47 C, 73.56; H, 8.32; N, 11.53

40 C16H22N2O C, 70.04; H, 8.08; N, 10.21 C, 69.78; H, 7.99; N, 10.34

41 C15H13N5O C, 64.51; H, 4.69; N, 25.07 C, 64.47; H, 4.39; N, 25.40

42 C15H13N5O C, 64.51; H, 4.69; N, 25.07 C, 64.39; H, 4.51; N, 24.93

43 C16H15N5O C, 65.52; H, 5.15; N, 23.88 C, 65.72; H, 5.07; N, 23.80

44 C15H21N5O C, 62.70; H, 7.37; N, 24.37 C, 62.76; H, 7.45; N, 24.12

45 C16H23N5O C, 63.76; H, 7.69; N, 23.24 C, 63.46; H, 7.58; N, 23.24


6.1.3. 5-Phenethyl-1H-tetrazole (23)The title compound was prepared from 3-phenylpropaneni-

trile (20) following the same procedure that was used for thesynthesis of 22. Yield 70%; mp 80e85 �C; IR 3134, 3029,2859, 2730, 2626, 1560, 1497, 1454, 1258, 1108,1050 cm�1; 1H NMR (300 MHz) d 3.15 (2H, t, J¼ 7.6 Hz),3.38 (2H, t, J¼ 7.6 Hz), 7.13e7.25 (5H, m); 13C NMR(75 MHz) d 25.29, 33.67, 126.72, 128.30, 128.67, 139.10,156.11. Anal. (C9H10N4) C, H, N.

6.1.4. N,N-Dimethyl-5-phenethyl-1H-tetrazole-1-carboxamide (3) and N,N-dimethyl-5-phenethyl-2H-tetrazole-2-carboxamide (5)

The title compounds were prepared from 23 following thesame procedure that was used for the synthesis of 1, 2 usinghexane/AcOEt¼ 1/1 as eluent for the chromatographic separa-tion of regioisomers. Compound 5 (less polar, 23%): oil; IR(CHCl3) 3009, 2941, 1740, 1498, 1393, 1311, 1252, 1168,1130, 1070 cm�1; 1H NMR (300 MHz) d 3.01 (3H, s), 3.22(3H, s), 3.11e3.34 (4H, m), 7.14e7.32 (5H, m); 13C NMR(75 MHz) d 27.12, 33.79, 38.05, 39.00, 126.39, 128.37,128.52, 140.09, 147.92, 165.53. Anal. (C12H15N5O) C, H, N.Compound 3 (more polar, 17%): oil; IR (CHCl3) 3014,2941, 1728, 1497, 1396, 1257, 1236, 1088, 1076 cm�1; 1HNMR (300 MHz) d 2.76 (3H, s), 3.09 (3H, s), 3.13 (2H, t,J¼ 7.6 Hz), 3.48 (2H, t, J¼ 7.6 Hz), 7.13e7.31 (5H, m);

13C NMR (75 MHz) d 25.65, 33.61, 37.58, 39.12, 126.68,128.58, 139.24, 147.84, 156.37. Anal. (C12H15N5O) C, H, N.

6.1.5. 5-(3-Phenylpropyl)-1H-tetrazole (24)The title compound was prepared from 4-phenylbutyroni-

trile (21) following the same procedure that was used for thesynthesis of 22. Yield 55%; mp 80e85 �C; IR 3130, 3024,2864, 2730, 2622, 1568, 1495, 1450, 1421, 1256, 1107,1047 cm�1; 1H NMR (300 MHz) d 2.19 (2H, m), 2.71 (2H,t, J¼ 7.5 Hz), 3.08 (2H, t, J¼ 7.7 Hz), 7.11e7.27 (5H, m);13C NMR (75 MHz) d 22.91, 29.03, 34.99, 126.25, 128.42,128.52, 140.54, 156.77. Anal. (C10H12N4) C, H, N.

6.1.6. N,N-Dimethyl-5-(3-phenylpropyl)-1H-tetrazole-1-carboxamide (4) and N,N-dimethyl-5-(3-phenylpropyl)-2H-tetrazole-2-carboxamide (6)

The title compounds were prepared from 24 following thesame procedure that was used for the synthesis of 1, 2 usingpreparative layer chromatography (silica gel, 0.5 cm thick)and hexane/acetone¼ 7/3 as eluent for the separation ofregioisomers. Compound 6 (less polar, 38%): oil; IR (CHCl3)3024, 2942, 2864, 1740, 1603, 1497, 1453, 1393, 1310,1167, 1071 cm�1; 1H NMR (300 MHz) d 2.16 (2H, m), 2.70(2H, t, J¼ 7.5 Hz), 2.99 (2H, t, J¼ 7.6 Hz) 3.09 (3H, s),3.24 (3H, s), 7.14e7.33 (5H, m); 13C NMR (75 MHz)d 24.76, 29.26, 35.11, 38.06, 39.10, 126.03, 128.42, 128.49,


141.19, 147.96, 166.14. Anal. (C13H17N5O) C, H, N. Com-pound 4 (more polar, 23%): oil; IR (CHCl3) 3024, 2942,2863, 1728, 1603, 1497, 1453, 1396, 1258, 1088 cm�1; 1HNMR (300 MHz) d 2.17 (2H, m), 2.74 (2H, t, J¼ 7.5 Hz),3.06 (5H, m), 3.20 (3H, s), 7.15e7.32 (5H, m); 13C NMR(75 MHz) d 23.59, 28.48, 35.02, 37.76, 39.33, 126.17,128.48, 140.80, 148.00, 156.89. Anal. (C13H17N5O) C, H, N.

6.1.7. 4-Azido-N-(cyanomethyl)-3-iodobenzamide (29)A solution of 28 [34] (7.250 g, 25.08 mmol) in SOCl2

(30 mL) was stirred at 100 �C for 2 h. Excess SOCl2 wasevaporated under vacuum and the residue of the acid chlo-ride (7.711 g, 100%) was dissolved in dry CH2Cl2 (50 mL),and H2NCH2CN$H2SO4 (4.253 g, 27.59 mmol) and Et3N(17.6 mL, 125.71 mmol) were added under stirring at 0 �C.The mixture was stirred at room temperature for 15 h, dilutedwith water, and extracted with AcOEt. The organic phase waswashed with 2 N HCl, water until neutral, 2 N NaOH, and wa-ter until neutral, dried (Na2SO4), and evaporated under vac-uum to leave a residue (5.239 g, 64%) of 29 as a whitesolid. Mp 65e68 �C; IR 3337, 3230, 3059, 3000, 2116,1643, 1588, 1538, 1474, 1298 cm�1; 1H NMR (300 MHz)d 4.38 (2H, d, J¼ 5.8 Hz), 6.81 (1H, m), 7.18 (1H, d,J¼ 8.4 Hz), 7.83 (1H, dd, J¼ 8.4, 2.0 Hz), 8.21 (1H, d,J¼ 2.0 Hz); 13C NMR (75 MHz) d 28.15, 87.68, 115.87,118.28, 128.66, 130.18, 139.11, 146.07, 165.06. Anal.(C9H6IN5O) C, H, N.

6.1.8. N-((1H-Tetrazol-5-yl)methyl)-4-azido-3-iodobenzamide (30)

The title compound was prepared from 29 following thesame procedure that was used for the synthesis of 22. Yield(39%); mp 188e190 �C; IR 3287, 2855, 2125, 1636, 1447,1301, 1035 cm�1; 1H NMR (300 MHz, DMSO-d6) d 3.38(1H, br s), 4.76 (2H, s), 7.47 (1H, d, J¼ 8.1 Hz), 7.98 (1H,dd, J¼ 8.1, 1.8 Hz), 8.36 (1H, d, J¼ 1.8 Hz), 9.35 (1H, m);13C NMR (75 MHz, DMSO-d6) d 33.14, 33.24, 87.71,118.99, 129.00, 131.11, 138.42, 144.30, 154.48, 164.37,164.45. Anal. (C9H7IN8O) C, H, N.

6.1.9. 5-((4-Azido-3-iodobenzamido)methyl)-N,N-dimethyl-1H-tetrazole-1-carboxamide (7) and 5-((4-azido-3-iodobenzamido)methyl)-N,N-dimethyl-2H-tetrazole-2-carboxamide (8)

The title compounds were prepared from 30 following thesame procedure that was used for the synthesis of 1, 2 usinghexane/AcOEt¼ 45/55 as eluent for the chromatographic sep-aration of regioisomers. Compound 8 (less polar, 29%): mp125 �C; IR 3274, 2928, 2121, 1739, 1651, 1534, 1477, 1291,1085 cm�1; 1H NMR (300 MHz) d 3.17 (3H, s), 3.26 (3H,s), 4.99 (2H, d, J¼ 5.8 Hz), 7.10 (1H, d, J¼ 8.4 Hz), 7.81(1H, dd, J¼ 8.4, 2.2 Hz), 8.00 (1H, m), 8.20 (1H, d,J¼ 2.2 Hz); 13C NMR (75 MHz) d 34.22, 37.85, 39.38,87.33, 117.87, 128.32, 130.53, 138.99, 145.26, 147.73,154.49, 165.16. Anal. (C12H12IN9O2) C, H, N. Compound 7(more polar, 15%): mp 119 �C; IR 3279, 2933, 2123, 1735,1638, 1536, 1390, 1314, 1131 cm�1; 1H NMR (300 MHz)

d 3.13 (3H, s), 3.27 (3H, s), 4.97 (2H, d, J¼ 6.0 Hz), 7.13(1H, d, J¼ 8.4 Hz), 7.48 (1H, m), 7.86 (1H, dd, J¼ 8.4,2.1 Hz), 8.25 (1H, d, J¼ 2.1 Hz); 13C NMR (75 MHz)d 35.25, 38.11, 39.13, 87.33, 117.87, 128.44, 131.02, 138.93,145.06, 147.39, 163.12, 165.06. Anal. (C12H12IN9O2) C, H, N.

6.1.10. N-(Cyanomethyl)biphenyl-3-carboxamide (36)To a stirred solution of biphenyl-3-carboxylic acid (31) [35]

(223 mg, 1.12 mmol) in DMF (4.5 mL) were added at 0 �CHOBt (181 mg, 1.18 mmol) and EDC (226 mg, 1.18 mmol).The mixture was stirred for 15 min at 0 �C and for 1 h atroom temperature, and H2NCH2CN$H2SO4 (207 mg,1.35 mmol) and Et3N (0.78 mL, 5.61 mmol) were added andthe mixture was stirred overnight at room temperature. Themixture was diluted with brine and extracted with AcOEt.The organic phase was washed with 2 N HCl solution, brine,saturated NaHCO3 and brine, dried (Na2SO4), and evaporatedunder vacuum. The residue (349 mg) was chromatographed onsilica gel (11 g) using CH2Cl2/AcOEt¼ 95/5 as eluent to give224 mg (85%) of 36 as an oil; IR (CHCl3) 3453, 3339, 3065,2943, 1676, 1509, 1476, 1304, 1257, 1202, 1145, 1091 cm�1;1H NMR (300 MHz) d 4.31 (2H, d, J¼ 5.6 Hz), 7.30e7.99(10H, m); 13C NMR (75 MHz) d 28.10, 116.36, 125.93,126.06, 127.09, 127.91, 128.94, 129.23, 131.01, 133.06,139.79, 141.83, 167.82. Anal. (C15H12N2O) C, H, N.

6.1.11. N-((1H-Tetrazol-5-yl)methyl)biphenyl-3-carboxamide (41)

The title compound was prepared from 36 following thesame procedure that was used for the synthesis of 22. Yield86%; oil; IR (CHCl3) 3410, 3338, 3031, 2960, 2925, 1656,1518, 1476, 1306, 1225, 1088 cm�1; 1H NMR (300 MHz,CDCl3/CD3OD¼ 9/1) d 4.86 (2H, s), 7.32e8.05 (9H, m);13C NMR (75 MHz, CDCl3/CD3OD¼ 9/1) d 33.62, 126.01,126.12, 127.13, 127.17, 127.85, 128.95, 129.10, 130.66,133.84, 140.07, 141.70, 168.77. Anal. (C15H13N5O) C, H, N.

6.1.12. 5-(Biphenyl-3-ylcarboxamidomethyl)-N,N-dimethyl-1H-tetrazole-1-carboxamide (9) and 5-(biphenyl-3-ylcarboxamidomethyl)-N,N-dimethyl-2H-tetrazole-2-carboxamide (14)

The title compounds were prepared from 41 following thesame procedure that was used for the synthesis of 1, 2 usinghexane/acetone¼ 65/35 as eluent for the chromatographicseparation of regioisomers. Compound 14 (less polar, 14%):oil; IR (CHCl3) 3451, 3033, 2944, 1731, 1667, 1510, 1396,1252, 1091 cm�1; 1H NMR (300 MHz) d 3.10 (3H, s), 3.20(3H, s), 5.03 (2H, d, J¼ 5.8 Hz), 7.27e8.02 (10H, m); 13CNMR (75 MHz) d 34.34, 38.61, 39.47, 125.82, 126.02,127.16, 127.83, 128.91, 129.11, 130.71, 133.65, 140.02,141.82, 148.06, 154.55, 167.70. Anal. (C18H18N6O2) C, H,N. Compound 9 (more polar, 10%): oil; IR (CHCl3) 3452,3414, 3022, 2941, 1746, 1675, 1509, 1392, 1251, 1072,1000 cm�1; 1H NMR (300 MHz) d 3.09 (3H, s), 3.23 (3H,s), 5.03 (2H, d, J¼ 5.6 Hz), 7.26e8.02 (10H, m); 13C NMR(75 MHz) d 35.42, 38.11, 39.20, 125.97, 126.28, 127.18,


127.77, 128.88, 129.05, 130.54, 134.28, 140.14, 141.78,163.42, 167.66. Anal. (C18H18N6O2) C, H, N.

6.1.13. N-(Cyanomethyl)biphenyl-4-carboxamide (37)The title compound was prepared from acid 32 following

the same procedure that was used for the synthesis of 36. Yield76%; mp 197e198 �C; IR 3307, 2923, 1645, 1608, 1531,1484, 1413, 1309 cm�1; 1H NMR (300 MHz, CDCl3/CD3OD¼ 1/1) d 4.32 (2H, d, J¼ 8.2 Hz), 7.38e7.95 (9H,m); 13C NMR (75 MHz, CDCl3/CD3OD¼ 1/1) d 28.02,116.69, 127.38, 127.45, 128.20, 128.39, 129.17, 131.58,140.03, 145.34, 168.50. Anal. (C15H12N2O) C, H, N.

6.1.14. N-((1H-Tetrazol-5-yl)methyl)biphenyl-4-carboxamide (42)

The title compound was prepared from 37 following thesame procedure that was used for the synthesis of 22. Yield67%; mp 275e276 �C; IR 3288, 2856, 1638, 1532, 1484,1433, 1310, 1246, 1222, 1054 cm�1; 1H NMR (300 MHz,DMSO-d6) d 3.28 (1H, br s), 4.49 (2H, d, J¼ 5.6 Hz),7.07e7.73 (9H, m), 9.02 (1H, t, J¼ 5.6 Hz); 13C NMR(75 MHz, DMSO-d6) d 33.23, 126.44, 126.78, 128.02,128.93, 131.45, 132.31, 139.00, 143.02, 154.72, 166.26.Anal. (C15H13N5O) C, H, N.

6.1.15. 5-(Biphenyl-4-ylcarboxamidomethyl)-N,N-dimethyl-2H-tetrazole-2-carboxamide (15)

The title compound was prepared from 42 following thesame procedure that was used for the synthesis of 1, 2 usinghexane/AcOEt¼ 45/55 as eluent for the chromatographic sep-aration of regioisomers. Attempts to isolate 1,5-regioisomer 10in a pure form failed owing to its propensity to isomerise.Compound 15 (49%): mp 186e188 �C; IR 3289, 2857,1725, 1627, 1552, 1487, 1397, 1307, 1249, 1084 cm�1; 1HNMR (300 MHz, DMSO-d6) d 2.91 (3H, s), 3.08 (3H, s),4.90 (2H, d, J¼ 5.4 Hz), 6.88 (1H, br s), 7.41e8.00 (9H,m); 13C NMR (75 MHz, DMSO-d6) d 30.24, 33.36, 36.91,124.69, 126.44, 126.69, 127.81, 128.82, 131.77, 138.86,143.09, 147.31, 151.28, 154.17. Anal. (C18H18N6O2) C, H, N.

6.1.16. 2-(Biphenyl-4-yl)-N-(cyanomethyl)acetamide (38)The title compound was prepared from 2-(biphenyl-4-yl)-

acetic acid (33) following the same procedure that was usedfor the synthesis of 36. Yield 69%; mp 146e147 �C; IR(KBr) 3249, 3046, 1683, 1644, 1541, 1486, 1407, 1256,1160, 1029 cm�1; 1H NMR (300 MHz, CD3OD) d 3.61 (2H,s), 4.43 (2H, s), 7.34e7.56 (9H, m); 13C NMR (75 MHz,CD3OD) d 27.62, 42.43, 116.53, 127.24, 127.65, 127.71,129.09, 129.88, 133.65, 140.56, 140.95, 172.80. Anal.(C16H14N2O) C, H, N.

6.1.17. N-((1H-Tetrazol-5-yl)methyl)-2-(biphenyl-4-yl)acetamide (43)

The title compound was prepared from 38 following thesame procedure that was used for the synthesis of 22. Yield67%; mp 187e189 �C; IR 3334, 3034, 1646, 1548, 1517,1487, 1423, 1261, 1231, 1160, 1076 cm�1; 1H NMR

(300 MHz, DMSO-d6) d 3.60 (2H, s), 4.09 (2H, m), 7.30e7.59 (9H, m); 13C NMR (75 MHz, DMSO-d6) d 27.53,42.42, 116.36, 127.13, 127.56, 127.66, 128.97, 129.81,133.36, 140.51, 140.73, 172.26. Anal. (C16H15N5O) C, H, N.

6.1.18. 5-((2-(Biphenyl-4-yl)acetamido)methyl)-N,N-dimethyl-1H-tetrazole-1-carboxamide (11) and 5-((2-(biphenyl-4-yl)acetamido)methyl)-N,N-dimethyl-2H-tetrazole-2-carboxamide (16)

The title compounds were obtained as an inseparable 0.82:1mixture from 43 following the same procedure that was usedfor the synthesis of 1, 2. Yield 31%; IR (CHCl3) 3433,3009, 2942, 1740, 1676, 1504, 1489, 1395, 1257, 1165,1064 cm�1; 1H NMR (300 MHz) d 3.01 (3H, s), 3.14 and3.19 (3H, 2s), 3.60 and 3.66 (2H, 2s), 4.76 and 4.77 (2H,2d, J¼ 5.8 Hz), 6.72 and 7.03 (1H, 2m), 7.26e7.58 (9H,m); 13C NMR (75 MHz) d 33.96, 35.00, 37.83, 38.11, 39.10,39.32, 42.73, 42.97, 126.98, 127.38, 127.41, 127.57, 128.81,129.79, 129.88, 133.37, 133.57, 140.23, 140.26, 140.47,140.55, 147.56, 147.81, 154.46, 163.25, 171.27, 171.60.

6.1.19. N-(Cyanomethyl)-7-phenylheptanamide (39)The title compound was prepared from 7-phenylheptanoic

acid (34) following the same procedure that was used for thesynthesis of 36 using hexane/AcOEt¼ 7/3 as eluent for thechromatographic purification. Yield 61%; oil; IR (CHCl3)3452, 3318, 3034, 2934, 2858, 1686, 1602, 1501, 1414,1234, 1204 cm�1; 1H NMR (300 MHz) d 1.31e1.36 (4H,m), 1.61 (4H, m) 2.21 (2H, t, J¼ 7.4 Hz), 2.59 (2H, t,J¼ 7.6 Hz), 4.10 (2H, d, J¼ 5.8 Hz), 6.60 (1H, m), 7.14e7.30 (5H, m); 13C NMR (75 MHz) d 25.22, 27.41, 28.86,28.98, 31.20, 35.81, 35.88, 116.33, 125.64, 128.25, 128.38,142.59, 173.57. Anal. (C15H20N2O) C, H, N.

6.1.20. N-((1H-Tetrazol-5-yl)methyl)-7-phenylheptanamide(44)

The title compound was prepared from 39 following thesame procedure that was used for the synthesis of 22. Yield56%; mp 124e132 �C; IR 3297, 3025, 2934, 2854, 1659,1521, 1434, 1333, 1250, 1235, 1080, 1053 cm�1; 1H NMR(300 MHz, CDCl3/CD3OD¼ 9/1) d 1.27e1.38 (4H, m),1.52e1.66 (4H, m) 2.21 (2H, t, J¼ 7.6 Hz), 2.58 (2H, t,J¼ 7.5 Hz), 4.66 (2H, s), 7.11e7.31 (5H, m); 13C NMR(75 MHz, CDCl3/CD3OD¼ 9/1) d 25.46, 28.95, 29.11,31.27, 32.71, 35.89, 36.06, 125.69, 128.31, 128.43, 142.69,154.76, 175.12. Anal. (C15H21N5O) C, H, N.

6.1.21. N,N-Dimethyl-5-((7-phenylheptanamido)methyl)-1H-tetrazole-1-carboxamide (12) and N,N-dimethyl-5-((7-phenylheptanamido)methyl)-2H-tetrazole-2-carboxamide(17)

The title compounds were prepared from 44 following thesame procedure that was used for the synthesis of 1, 2 usinghexane/acetone¼ 65/35 as eluent for the chromatographicseparation of regioisomers. Compound 17 (less polar, 38%):mp 74e76 �C; IR 3310, 3062, 3027, 2926, 2853, 1727,1653, 1542, 1420, 1397, 1251, 1087 cm�1; 1H NMR


(300 MHz) d 1.27e1.35 (4H, m), 1.52e1.62 (4H, m) 2.21(2H, t, J¼ 7.5 Hz), 2.58 (2H, t, J¼ 7.6 Hz), 3.10 (3H, s),3.22 (3H, s), 4.80 (2H, d, J¼ 5.8 Hz), 6.94 (1H, m), 7.13e7.30 (5H, m); 13C NMR (75 MHz) d 25.38, 28.88, 29.03,31.18, 33.69, 35.82, 36.09, 37.91, 39.42, 125.62, 128.24,128.38, 142.63, 147.91, 154.65, 173.72. Anal. (C18H26N6O2)C, H, N. Compound 12 (more polar, 26%): mp 55e57 �C;IR 3312, 3066, 3029, 2937, 2846, 1732, 1651, 1548, 1410,1383, 1277, 1215, 1134, 1073 cm�1; 1H NMR (300 MHz)d 1.26e1.39 (4H, m), 1.54e1.69 (4H, m) 2.26 (2H, t,J¼ 7.5 Hz), 2.60 (2H, t, J¼ 7.7 Hz), 3.09 (3H, s), 3.24 (3H,s), 4.80 (2H, d, J¼ 5.6 Hz), 6.40 (1H, m), 7.13e7.31 (5H,m); 13C NMR (75 MHz) d 25.47, 28.95, 29.09, 31.25, 34.87,35.87, 36.38, 38.17, 39.17, 125.63, 128.26, 128.41, 142.70,147.61, 163.46, 173.31. Anal. (C18H26N6O2) C, H, N.

6.1.22. N-(Cyanomethyl)-8-phenyloctanamide (40)The title compound was prepared from 8-phenyloctanoic

acid (35) following the same procedure that was used for thesynthesis of 36 using hexane/AcOEt¼ 7/3 as eluent for thechromatographic purification. Yield 47%; mp 39e40 �C; IR(CHCl3) 3452, 3323, 3034, 2932, 2857, 2337, 1686, 1602,1498, 1453, 1348, 1263, 1263, 1206, 1031 cm�1; 1H NMR(300 MHz) d 1.30 (6H, m), 1.59 (4H, m), 2.20 (2H, t,J¼ 7.5 Hz), 2.57 (2H, t, J¼ 7.6 Hz), 4.10 (2H, d,J¼ 5.8 Hz), 6.52 (1H, m), 7.11e7.30 (5H, m); 13C NMR(75 MHz) d 25.30, 27.42, 29.08, 29.13, 31.34, 35.90, 35.95,116.30, 125.62, 128.26, 128.39, 142.76, 173.54. Anal.(C16H22N2O) C, H, N.

6.1.23. N-((1H-Tetrazol-5-yl)methyl)-8-phenyloctanamide(45)

The title compound was prepared from 40 following thesame procedure that was used for the synthesis of 22. Yield79%; mp 118e120 �C; IR 3291, 3024, 2930, 2852, 1659,1526, 1433, 1333, 1251, 1231, 1083, 1053 cm�1; 1H NMR(300 MHz, CDCl3/CD3OD¼ 9/1) d 1.29 (6H, m), 1.57e1.61(4H, m) 2.26 (2H, t, J¼ 7.6 Hz), 2.58 (2H, t, J¼ 7.7 Hz),4.70 (2H, m), 7.13e7.31 (5H, m), 7.67 (1H, m); 13C NMR(75 MHz, CDCl3/CD3OD¼ 9/1) d 25.48, 29.11, 31.35,32.46, 32.57, 35.92, 36.12, 36.16, 125.63, 128.27, 128.40,142.76, 153.20, 175.43. Anal. (C16H23N5O) C, H, N.

6.1.24. N,N-Dimethyl-5-((7-phenyloctanamido)methyl)-1H-tetrazole-1-carboxamide (13) and N,N-dimethyl-5-((7-phenyloctanamido)methyl)-2H-tetrazole-2-carboxamide(18)

The title compounds were prepared from 45 following thesame procedure that was used for the synthesis of 1, 2 usingpreparative layer chromatography (silica gel, 0.5 cm thick)and hexane/acetone¼ 60/40 as eluent for the chromatographicseparation of regioisomers. Compound 18 (less polar, 38%):mp 74e76 �C; IR 3302, 3074, 2927, 2852, 1728, 1655,1548, 1413, 1397, 1334, 1251, 1123, 1087 cm�1; 1H NMR(300 MHz) d 1.29 (6H, m), 1.55e1.61 (4H, m) 2.21 (2H, t,J¼ 7.6 Hz), 2.58 (2H, t, J¼ 7.7 Hz), 3.09 (3H, s), 3.22 (3H,s), 4.81 (2H, d, J¼ 6.0 Hz), 6.95 (1H, m), 7.14e7.30 (5H,

m); 13C NMR (75 MHz) d 25.45, 29.07, 29.13, 31.36, 33.6935.90, 36.12, 37.90, 39.42, 125.58, 128.22, 128.37, 142.75,147.91, 154.64, 173.75. Anal. (C19H28N6O2) C, H, N. Com-pound 13 (more polar, 26%): mp 55e57 �C; IR 3294, 3068,2927, 2849, 1732, 1655, 1549, 1490, 1384, 1300, 1248,1133, 1075 cm�1; 1H NMR (300 MHz) d 1.32 (6H, m),1.61e1.68 (4H, m) 2.26 (2H, t, J¼ 7.6 Hz), 2.59 (2H, t,J¼ 7.6 Hz), 3.09 (3H, s), 3.25 (3H, s), 4.81 (2H, d,J¼ 5.8 Hz), 6.37 (1H, m), 7.12e7.30 (5H, m); 13C NMR(75 MHz) d 25.51, 29.11, 29.18, 31.38, 34.86, 35.93, 36.41,38.17, 39.18, 125.58, 128.23, 128.39, 142.79, 147.59,163.42, 173.30. Anal. (C19H28N6O2) C, H, N.

6.2. X-ray structure determination of tetrazole 2

X-ray diffraction data for compound 2 (slow crystallisationfrom CH2Cl2/hexane) were collected on a Rigaku AFC5R dif-fractometer at 298 K, using graphite-monochromated Cu Karadiation (l¼ 1.54178 A) and a rotating anode generator.The structure was solved by direct methods and refined byfull-matrix least-squares with anisotropic thermal parameters,using the SIR2004 structure determination package [39]. Thehydrogen atoms were idealised (CeH¼ 0.96 A). Each H atomwas assigned the equivalent isotropic temperature factor ofthe parent atom and allowed to ride it. The final differenceFourier map with a root-mean-square deviation of electrondensity of 0.64 eA�3 showed no significant features.

6.3. Biological evaluation

6.3.1. Assay of AEA cellular reuptakeThe effect of compounds on the uptake of [14C]anandamide

by rat basophilic leukemia (RBL-2H3) cells was studied by us-ing 2.4 mM (10,000 cpm) of [14C]anandamide [40]. Themethod consisted of incubating the cells with [14C]ananda-mide for 90 s at 37 �C, in the presence or absence of varyingconcentrations of the inhibitors, which were administered tocells 10 min prior to incubation with [14C]anandamide. In allcases, residual [14C]anandamide in the incubation medium af-ter extraction with CHCl3/CH3OH¼ 2/1 (by volume), deter-mined by scintillation counting of the lyophilized organicphase, was used as a measure of the amount of anandamidethat was taken up by cells. In some cases, the formation of[14C]ethanolamine from [14C]anandamide was measured bycounting the water phase of the incubation medium extract.Data are expressed as the concentration exerting 50% inhibi-tion of anandamide uptake (IC50) calculated by GraphPad�.Non-specific binding of [14C]anandamide to cells and plasticdishes was determined in the presence of 100 mM anandamideand was never higher than 40% of total uptake.

6.3.2. Assay of fatty acid amide hydrolase (FAAH)The effect of compounds on the enzymatic hydrolysis of

anandamide was studied as described previously [40] usingmembranes prepared from either rat brain, incubated with thetest compounds and [14C]anandamide (2.4 mM) in 50 mMtriseHCl, pH 9, for 30 min at 37 �C. [14C]ethanolamine


produced from [14C]anandamide hydrolysis was measured byscintillation counting of the aqueous phase after extraction ofthe incubation mixture with 2 volumes of CHCl3/CH3OH¼ 2/1(by volume). With active compounds, data are expressed asthe concentration exerting 50% inhibition of AEA hydrolysis(IC50), calculated by GraphPad.

6.3.3. Assay of DAGL activityConfluent COS cells, overexpressing DAGLa, were har-

vested in triseHCl buffer, pH 7 and homogenized in a homog-enizer (Dounce). The homogenates were centrifuged at 4 �C at800 g (5 min) and then at 10 000 g (25 min). The 10 000 gmembrane fraction was incubated in incubation buffer (triseHCl 50 mM and CaCl2 1 mM) at pH 7.0 at 37 �C for20 min, with synthetic sn-1-[14C]oleoyl-2-arachidonoyl-glyc-erol (1.0 mCi/mmol, 25 mM). After the incubation, lipidswere extracted with 2 volumes of CHCl3/MeOH¼ 2/1 (byvolume), and the extracts were lyophilized under vacuum. Ex-tracts were fractionated by TLC on silica on plastic plates us-ing CHCl3/MeOH/NH4OH (85/15/1 by volume) as the elutingsystem. Bands corresponding to [14C]oleic acid were cut, andtheir radioactivity was counted with a b-counter.

6.3.4. Assay of MAGL activityThe 10 000 g cytosolic fraction from COS cells was incu-

bated in triseHCl 50 mM, at pH 7.0 at 37 �C for 20 min,with synthetic 2-[3H] arachidonoyl-glycerol (1.0 mCi/mmol,25 mM). After the incubation, lipids were extracted with2 volumes of chloroform/methanol¼ 2/1 (by volume), andthe extracts were lyophilized under vacuum. Extracts werefractionated by TLC on silica on plastic plates using CHCl3/MeOH/NH4OH (85/15/1 by volume) as the eluting system.Bands corresponding to [3H]arachidonic acid were cut, andtheir radioactivity was counted with a b-counter.

Acknowledgements

The authors are grateful to Mr. M. Allara for valuable tech-nical assistance.

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carbamoyl tetrazoles as inhibitors of endocannabinoid inactivation: a critical revisitation

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